1. Field of the Invention
The present invention is relates to a method for the in vitro aseptic mass production and sporulation of arbuscular mycorrhizal fungi, especially cultivating the fungal propagules and spores by using root organs of symbiotic host plant in a limited aseptic space where liquid nutrient medium steadily floods and then drains out at regular intervals.
2. Description of Related Art
After well development of modern science and technology, the abusiveness of chemical fertilizers and pesticides in current agriculture and horticulture causes pollution and destruction of natural environment even though output of crops rises rapidly. Therefore, governments of different countries in the world all give highly regards to development and management of sustainable agriculture in recent years. And one feasible way in accord with the sustainable agricultural issues is to generalize the recyclable organic agricultural cultivation methods that are treated as the most remarkable ones. Among these methods, utilizing naturally existent plant growth prompting rhizomicrobes (PGPR) to enhance absorption of aliments and resistance against a hostile circumstance is an influential developing orientation in both of domestic and overseas researches
Arbuscular mycorrhizal fungi (AMF), being a kind of beneficial symbiotic fungi, can come into symbiosis with virtually 70% of all cultivated plants by infecting their roots and colonizing invasively inside root cells, which also makes them getting the name of endomycorrhizal fungi. They are mainly characterized by their tiny, bifurcate and branched arbuscules grown in cortex cells of plant roots whereby they are named. Hyphae of fungal mycorrhizae will extend outwards from the root surface functioning as root fibrils after the mycorrhizae is grown, and thus contribute to expansion of spreading range of the root system to promote root capabilities of nutrient uptake. Mycorrhizal fungi is proofed to be a kind of microbial fertilizers due to their stimulating actions on plant growth which is confirmed by many domestic or overseas research reports. The actions include: (1) increasing absorption of microelements; (2) increasing absorption of phosphates; (3) promoting resistance of infected plants to pathogens; (4) enhancing capability of drought resistance; (5) suppressing absorption of nocuous elements; and (6) connecting with other plants via hyphae.
Accordingly, to efficiently utilize the above-mentioned microorganism can not only enhance the growth of plants but also lower opportunities of using fertilizers and agricultural chemicals to further contribute to environment protection. However, the absolute symbiosis between AMF and plants, i.e. no AMF propagules or spores have been successfully cultivated in a sterile medium without a host plant despite the large amount of domestic and overseas researches carried out to date. The most difficult reason to apply AMF as one of microorganism fertilizers is no cheaper and more efficient method that is found now to mass-produce AMF inoculums of a high quality without any adulterated microbial contaminants for commercial applications. Therefore the price is still high enough to affect the farmer willing of adopting the above-mentioned fertilizer. It will be very helpful for generalizing this excellent microbial fertilizer to know in advance how to lower the cost of using AMF inoculums.
Conventional methods to cultivate AMF propagules include as follows:
1. Potted cultivating method carried out with traditional potted plants having AMF inoculation which is easily understood from the previous description is first preparing some mycorrhizae or spores of AMF as inoculums to be inoculated into the radicle of a host plant cultivated in pots or a greenhouse and then cultivating AMF therein. However, the time required for cultivation of AMF is quite long and the product is unexpectable due to the possibility of introducing contaminants in the soil at the same time.
2. Hydroponic cultivating method is to cultivate symbiotic host plants inoculated with AMF in a specialized hydroponic device with nutrient liquid in which the host plants and AMF inoculums therein are submerged to enhance the growth of mycorrhizae and sporulation of AMF. However, though efficiency of AMF production for this method is better than the method used for potted plants, frequently refreshing the nutrient liquid is needed to prevent from serious problem of adulterated microbial contaminants. And the soaked state of the host plants and AMF therein caused by aquatic environment in this method is not the normally and naturally growth condition for AMF which results in the quantity of AMF sporulation failing to increase.
3. Aeroponic cultivating method is to cultivating symbiotic host plants inoculated with AMF in a specialized aeroponic container where vaporized nutrient liquid is provided to the plants and AMF therein. Cultivating large quantities of AMF propagules becomes possible even in the soilless situation. But the deficiencies of this method comprise the need of building a specific aeroponic container, the requirement of huge amount of vaporized nutrient liquid with necessary work to watch and refresh them, and failure to avoid the problem of adulterated microbial and pathogen contaminants.
4. Transformed root organ cultivating method uses isolated plant roots genetically transformed by the Ri plasmid of Agrobacterium rhizogenes (Tepfer, 1984, Cell, Vol. 37, 959-967) to be able to grow rapidly and independently and be inoculated with pure AMF to become a symbiotic root system (Mugnier and Mosse, 1987, Phytopathology, Vol. 77, 1045-1050). It is an advanced cultivating method to acquire AMF propagules without adulterated microbial contaminants in specialized cultivating circumstances after pure AMF are inoculated into the transformed root organ.
Mugnier et al. in U.S. Pat. No. 4,599,312 issued on Jul. 8, 1986 in the name of Rhone-Poulenc Agrochimie, Wood et al. in European Patent Application No. EP-A-209,627 published on Jan. 28, 1987, and Fortin et al. in U.S. Pat. No. 5,554,530 issued on Sep. 10, 1996 in the name of Univerfsite de Montreal all teach cultivating transformed root organs and AMF therein by adopting the solidified medium in the transformed root organ cultivating method. However, root organs have to penetrate themselves into the solidified medium used to cultivate root organs aseptic in vitro to dissolve nutrients from the solidified medium, and it is hard to the root organs for growth that the solidified medium is oxygen-poor environment. Accordingly a plane type cultivating is the only way to do because the growth of the root system is hindered by the solid medium. Comparing to the potted, hydroponic and aeroponic cultivating methods that all practice the solid type cultivating using the complete plant root system, the adoption of solidified medium is not integrally efficient enough and needs much more procedures in the regaining process by first liquefying the solidified medium and then filtering the liquefied solution to make final products. It costs high for mass production and can not proceed on a large scale.
The transformed root organ cultivating method can use liquid (solution) medium to cultivate the root system too. Three types including the submerged style, rotating drum, and the airlift style are described as followed:
The submerged style indicates cultivating transformed root organs in shallow liquid medium by still placed cultivation (Nuutila, 1995, Plant Cell Rep., Vol. 14, 505-509). In practice, the depth of the liquid nutrient medium cannot be large in order to prevent the root organs from the asphyxiant counteraction that is not good for growth. Therefore the submerged style is unable to contribute to the incremental reproduction of AMF propagules on a mass-produced scale.
Rotating drum, also called the vibrating style, indicates cultivating the root organs in a predetermined quantity of the liquid medium and adding the amount of dissolved oxygen in the solution by spinning stir or vibration. The stress caused by spinning stir and vibration will inhibit the growth of root organs and decrease the incremental reproduction rate of AMF at the same time.
The airlift style indicates cultivating root organs in a container full of liquid nutrient medium and releasing bubbles continuously from the bottom of the container to facilitate breathing of root organs (Jolicoeur, 1999, Biotechnology and Bioengineering, Vol. 63, No. 2, 224-232). The method which obviously overcomes the asphyxiant problem met in the submerged style and is free from the excess mechanical stress arising in the rotating drum style is a more feasible way in all methods adopting liquid medium. But sterile air should be injected persistently into the container, and the injection process usually costs high and allows for adulterated microbial contamination to occur. Meanwhile, both of the root organs and AMF therein are submerged continuously in the liquid medium during the process, and the liquid-full container is not a normal growth circumstance to the root organ and AMF though a large quantity of dissolved oxygen is obtainable in the container by injected air. Therefore the unit efficiency is lower than the submerged style.
In conclusion, the conventional difficulty of keeping low cost compatible with high quality of final products is always a chock point of cultivating AMF in vitro. The cheaper and lower technical methods of above-mentioned potted, hydroponic and aeroponic ones have the difficulty to control product quality and the risk of adulterated microbial contamination both discouraging farmers from being willing to adopt them. Higher quality can be maintained by using the forth method of transformed root organ which is the main worldwide struggling development. But the generalized range is limited due to the high cost of this method.
Therefore, how to overcome the problems met in the symbiotic cultivation of transformed root organs and AMF in order to lower cost, promote their production and maintain the steady quality of AMF species is in bad need and will become the main technological part to generalize this benefit microbial fertilizer.
Accordingly, one objective of the present invention is to provide a better circumstance to cultivate transformed root organs and AMF therein where effective provision of nutrients and well ventilation for oxygen are available to overcome the conventional negative influences of long-termed submergence and deficient ventilation on the root organs and AMF.
A second objective of the present invention is to provide a feasible and low-cost solution for the growth and sporulation of AMF cultivated in liquid aseptic symbiotic process by simplifying needed procedures, lowering the risk of contamination and promoting the incremental reproduction rate of AMF.
A third objective of the present invention is to provide a method which is controllable and adjustable to get the desired sporulation of AMF by changing different kinds of liquid medium or timing of the contact between AMF propagules and liquid medium to facilitate the mass production of a large amount of AMF propagules for generalizing this excellent microbial fertilizer to protect our living environment.
In order to achieve the objects set forth, a method for the in vitro aseptic mass production and sporulation of arbuscular mycorrhizal fungi is featured by providing symbiotic root organs with AMF propagules and spores after the root organs are inoculated with AMF inoculums in advance to form AMF propagules in the root organs and cultivating them in an aseptic container, and then providing the whole symbiotic root organs and AMF propagules with liquid medium for a temporary contact, then removing the liquid medium from the symbiotic root organs and arbuscular mycorrhizal fungal propagules and finally repeating former two steps periodically to facilitate the mass production and sporulation of the arbuscular mycorrhizal fungi.
Other objects, advantages and novel features of the invention will become more apparent from the following detailed description when taken in conjunction with the accompanying drawings.